Antigenic compounds

ABSTRACT

The present invention relates to novel antigenic compositions suitable for parenteral and mucosal administration, comprising an antigen within an aqueous medium and a carbohydrate, wherein the carbohydrate is in the form of a derivative which exhibits acidic moieties, preferably in the form of its carboxylic acid derivative. The antigenic compositions are in a stable solid form and, after reconstitution with a liquid excipient or adjuvant followed by administration through the parenteral or mucosal route, rapidly dissolve in the body fluids, thereby releasing the reconstituted vaccine into the body. Specifically, the lyophilized form may consist of a powder or a cake containing the antigen, preferably in an amorphous (glassy) state, which is formed from a liquid solution or suspension by drying or sublimation, preferably sublimation by lyophilisation.

The present invention relates to novel antigenic compositions suitablefor parenteral and mucosal administration, comprising an antigen withinan aqueous medium and a carbohydrate, wherein the carbohydrate is in theform of a derivative which exhibit acidic moieties, preferably in theform of its carboxylic acid derivative. The antigenic compositions arein a stable solid form and, after reconstitution with a liquid excipientor adjuvant followed by administration through the parenteral or mucosalroute, rapidly dissolve in the body fluids, thereby releasing thereconstituted vaccine into the body. Specifically, the lyophilised formmay consist of a powder or a cake containing the antigen, preferably inan amorphous (glassy) state, which is formed from a liquid solution orsuspension by drying or sublimation, preferably sublimation bylyophilisation.

There is currently a great deal of interest in the pharmaceutical areain formulations that ensure the stability of the protein during theprocessing and storage stages. Aqueous liquid formulations are theeasiest and more economical to handle during manufacturing, and are alsothe most convenient for the end-user. However chemical or physicaldegradations are commonly occurring phenomenon in liquid formulations,which lead to protein damages such as denaturation and degradation,often irreversible.

These problems can be overcome by using dried solids of stable proteinmixtures, particularly but not only of stable lyophilised proteinmixtures. Stable lyophilised formulations and the issues which may ariseduring their preparation have been described (Carpenter, J. 1997,Pharmaceutical Res. 14, 969-975). The formulation must be designed toensure protein stability, to respect the desired product configuration,the product tonicity, especially for parenteral administration, andpresent an elegant cake structure. Similar considerations apply to othersolid forms such as vacuum dried or spray-dried powders. Severalrequirements must be respected, an important one being to ensureisotonicity, critical for parenteral administration, especially when theproduct has a relatively low mass of protein per vial, as it is the casewith vaccine formulations, which usually contain around 50 μg of proteinper vial, on a total of around 15 mg. Salts, in particular NaCl, havebeen used to modify the tonicity of the mixture, but because itdecreases the freezing point of solutions it can make the formulationmore difficult to be frozen (due to a lower freezing point), and theelimination of water by sublimation will take more time. Polyalcoholsand and carbohydrates such as mannitol or lactose have also been usedwith success to achieve a strong cake while respecting the isotonicityof the mixture (M. C. Lai and E. M. Topp, J. of Pharm. Sci., 1999, 88,489-500).

One important problem arises when the lyophilisation process must becarried out on mixtures which contain a high salt concentration as it isthe case, for example, for proteins resulting from a purificationprocess carried out in the presence of high salt concentrations.Alternatively, in some cases proteins may need a high salt concentrationin order to remain in solution and/or to avoid aggregation andprecipitation. These problems are particularly of issue when the salt isNaCl, since NaCl when present in solution gives a relatively loweutectic melting and make a formulation difficult to lyophiliseproperly.

Several solutions have been proposed, but these are unsatisfactory. Theincrease in the carbohydrate concentrations is associated with i) anincrease in the osmotic pressure, ii) an incompatibility with parenteraladministration, and iii) problems with the reconstitution itself, forexample when the reconstitution liquid is already iso-osmotic by itself.Another alternative, the addition to the formulation of small quantitiesof mannitol, which cristallise easily and which leads to the formationof a sort of strong cristalline net in the cake (Kim, A. et al. J.Pharmaceutical Sc. 1998, 87, 931-935). does not apply to very high saltconcentrations such as concentrations of NaCl above 15 mM and certainlyabout 30 mM. The addition to the formulation of a high molecular weightpolymeric substance, unbiodegradable, as described by Taylor, L. andZographi G., J. Pharmaceutical Sc. 1998, 87, 1615-1621 may notcompatible with pharmaceutical compositions or vaccine safetyconsiderations.

The present invention offers an alternative to these formulations, whichdoes not suffer from the disadvantages listed above. The presentinvention resides in the finding that the provision of antigeniccompositions in a stable solid form is possible despite high saltconcentrations being present which would have normally hampered theconstitution of dried powders or of lyophilised cakes of acceptablestructure. In particular, the present invention resides in the findingof suitable excipients termed bulking agents compatible with drying orlyophilisation of antigen mixtures containing high NaCl concentrations,in particular containing NaCl concentration above 30 mM. The excipientsor bulking agents for use according to the present invention areadvantageous since, through the ionic charges that they carry, theyfavour and even increase, in the presence of high NaCl concentrations,the intermolecular interactions between the bulking agent molecules,thereby allowing the cake formation to take place and to remain stableover time.

The invention further provides an antigenic composition in solid formsuch as lyophilised, spray-dried or spray freeze-dried or vacuum driedform and in reconstituted liquid form. According to the invention thereis further provided a process for the preparation of a compositionaccording to the invention which comprises mixing the ingredients of thecomposition, and either freezing them and drying the frozen mixture, orspraying them (for example into warm air), or vacuum drying them, orspray freezing-drying.

In one embodiment, the solid forms of the present invention arelyophilised porous forms, termed “cakes”, or their ground form, whichare stable during processing and storage, which are compatible withparenteral administration and which have a low residual moisture. Thevaccine cakes of the present invention are formed from a liquid solutionor suspension of vaccine by sublimation, and in a preferred form of theinvention the sublimation is performed by lyophilisation.

In another embodiment the solid forms of the present invention are driedpowders, particularly spray-dried or spray freezing-drying orvacuum-dried powders which have a low residual moisture.

Accordingly, it is an object of the invention to provide novel antigeniccompositions suitable for freeze-drying, spray-drying or vacuum drying,comprising an antigen within an aqueous medium, a carbohydrate orderivative thereof which exhibits acidic moieties, and optionally apreservative. In a preferred embodiment, the aqueous antigenic mediumcontains a NaCl concentration of at least 15 mM, preferably of at least30 mM. Typically the aqueous antigenic solution or suspension will bebuffered, or pH adjusted to a pH compatible with parenteral or mucosaladministration. Preferably the solid forms include at least one antigenin an amount from about 0.00001% to about 5% by weight. More preferablythe antigen is in an amount of from about 0.001% to about 1% by weight.Still more preferably the antigen amount ranges from 0.01% to about0.05%. Typically the NaCl concentration of the solution will range fromabout 15 mM to about 150 mM. More preferably the NaCl concentration willbe at least 20 mM, most preferably at least about 30 mM. The bulkingmaterial will preferably be in an amount from about 50% to about99.99999% by weight, more preferably from 60% to about 90%.

The bulking agent according to the invention provides both chemical andphysical stability to the cake in the presence of high NaClconcentration and also prevents the cake to collapse. It also providesprotection to the antigen. The bulking agent is a carbohydrate orderivative thereof, which is preferably in amorphous or cristallinestate, preferably in amorphous state. A preferred carbohydrate accordingto the invention concerns a carbohydrate or derivative thereof wherehigh intermolecular interactions (as assessed for example by the meltingpoint of the carbohydrates, the higher the melting point of thecarbohydrate, the higher the intermolecular interactions in the crystal)can be obtained thereby allowing the solid form, in particular the cakestructure, to be more resistant to the collapse phenomenon during thelyophilisation. Preferably, high molecular weight carbohydrates areused.

Carbohydrate derivatives are preferred which exhibit a reactivefunction, such as an amino moiety or an acidic moiety for example.Accordingly this is preferred aspect of the invention to usecarbohydrate derivatives which exhibit acidic moieties, i.e. moietieswhich act as proton donors. The acidic moiety may be mono- orpoly-acidic. Specific acidic moieties which may be mentioned include—OP(O)(OH)₂ (phosphate ester), —O—P(O)—O—P(O)(OH)₂ (di phosphateester),), —O—P(O)—O—P(O)—O—P(O)(OH)₂ (triphosphate ester), —SO₄H(sulphate ester) and —CO₂H (carboxylic acid). Preferably the acidicmoiety is carboxylic acid. The carbohydrate derivative may optionally beused in conjunction with a carbohydrate, such as sucrose for example.

The carbohydrate or carbohydrate derivatives may optionally bemonohydrated, or polyhydrated (dihydrate, trihydrate, etc). In a stillpreferred embodiment, the carbohydrate is used in the form of itscarboxylic acid derivative. Preferably, the carbohydrate is used in theform of the salt derivative of the carboxylic acid itself. Accordinglyin one aspect the invention provides antigenic compositions suitable forfreeze-drying, spray-drying spray freeze drying or vacuum drying,comprising an antigen within an aqueous medium, a carbohydrate, andoptionally a preservative, wherein the carbohydrate is in the form ofits carboxylic acid derivative.

Most preferably the carbohydrates derivatives are used in the form oftheir salt, preferably the calcium or sodium salt, most preferably theirsodium salt.

Non-limiting examples of suitable carbohydrates fulfilling the purposesof the invention include the carboxylic acid derivatives of thefollowing: sucrose, maltose; trehalose, raffinose, lactose, fructose,galactose, mannose, maltulose, iso-maltulose and lactulose, or dextrose.Preferred bulking agents are the carboxylic acid derivatives oflactitol, sorbitol, glucose, galactose and galactitol, namelylactobionic acid, gluconic acid, glucuronic acid, galacturonic acid, andgalactaric acid, respectively. These may be in their non hydrated orhydrated form (see Table 1). Even more preferably, lactobionic acid(pictured in FIG. 1), glucuronic acid, gluconic acid and lactobionicacid are used, most preferably in a salt form favouring sugar-sugarintermolecular interactions. Sodium or calcium salts are preferred. Themost preferred bulking agent is sodium lactobionate. Typically thecarboxylic acid carbohydrate derivatives are used at a concentration (ofthe formulated liquid mix before lyophilisation) of about 80 mM andabove. This provides an acceptable cake structure and ensure stabilityof the structre over time.

Table 1 shows that in the crystallin solid state domain, molecule tomolecule interactions are higher (as evidenced by a higher meltingpoint) for ionic molecules compared to the corresponding non ionicmolecules. TABLE 1 Carbohydrates and their carboxylic and saltderivatives Carbohydrate & Melting derivatives Characteristics pointsLactitol 4-O-β-D-galactopyranosyl-D-glucitol 146° C. Lactobionic acid4-O-β-D-galactopyranosyl-D-gluconic acid 195° C. A lactitol structurewith one of the primary alcohol of the glucitol part replaced by acarboxylic acid; thus creating an opportunity for ionic bond interactionSorbitol 1,2,3,4,5,6-hexanehexol 110° C. or D-glucitol Gluconic acid2,3,4,5,6-pentahydroxy-hexanoic acid 131° C. A glucitol structure with aprimary alcohol replaced by a carboxylic acid; thus creating an ionicbond interaction Ammonium The ammonium salt of gluconic acid 154° C.gluconat Glucose α-D-glucopyranose α: 146° C., β: 148° C. Glucuronicacid A glucose with the primary alcohol replaced by a 165° C. carboxylicacid; thus creating an ionic bond interaction Galactoseα-D-galactopyranose 118° C. monohydrate Galacturonic acid A galactosewith the primary alcohol replaced by α: 159° C., monhydrate a carboxylicacid; thus creating an ionic bond β: 166° C. interaction Galactitol1,2,3,4,5,6-hexanehexol 188° C. Galactaric acid A galactitol with twoprimary alcohols replaced 225° C. by two carboxylic acids; thus creatingtwo ionic bond interactions

By acceptable cake stucture is meant any, and preferably all of thefollowing criteria:

-   -   a cake volume similar to the volume of the starting liquid prior        to the lyophilisation step;    -   a cake having similar sizes measurement compared to the liquid        prior to the lyophilisation step. In other words, a cake exibing        as less retraction as possible in sizes measument compared to        the initial liquid before the lyophilisation step;    -   a cake showing visual homogeous aspect.

The cake size and volume measurement is made according to the proceduredescribed in the Example section, under paragraph 3.2.

Furthermore, an acceptable cake structure is also understood to be acake which does not show any change in its shape, size and color afterhaving undergone an accelerated stability test during one week at 37°C., or during real time stability for a period of at least 6 months,preferably of at least one year, and ideally of at least two years.

The preservative is selected from antioxidants, free radicals scavengersand reducing agents. Preferred are antioxidants such as alkyl gallates,butylated hydroxyanisole, butylated hydroxytoluene, nordihydroguaiareticacid and the tocopherols, ascorbic acid, isoascorbic acid, potassium andsodium salts of sulfurous acid, sodium formaldehyde sulphoxylate, citricacid, edetic acid and its salts, lecithin, tartaric acid, thiodiproionicacid.

The process for preparing the antigenic composition according to thepresent invention is suitably carried out using any lyophilisation,vacuum drying or spray drying, spray freezing drying technique commonlyused within the pharmaceutical area. For example, the technique oflyophilisation, and details of other suitable excipients, may be foundin Cameron et al., “Good Pharmaceutical freeze-drying Practice”,Interpharm, Buffalo Grove (1997). Accordingly, in a further aspect theinvention provides a process for preparing an antigenic composition asdefined herein which comprises mixing the NaCl-comprising antigensolution, the bulking agent, and optionally the buffer and thepreservative, and subjecting the mixture to a lyophilisation, vacuumdrying or spray drying spray freezing drying procedure. A preferredprocess according to the invention is a vial freeze-drying processalthough ambient temperature evaporation is encompassed within thepresent invention. Such a process comprises aseptic filling, in sterilevials, of a sterile filtered solution of the antigenic composition,followed by sublimation by lyophilisation. A sterile freeze-dryingrubber stopper is partially inserted into the vial, then the vial isfrozen, e.g. at a temperature from −195.8° C. to −5° C., preferablybetween −80° C. and −10° C., and thereafter vacuum-dried in the frozenstate. After drying the stopper is fully inserted before removing thevial from the lyophilisation unit.

A preferred process according to the invention is a spray-drying processin which tiny droplets of the composition are obtained by pumping theliquid composition in a spray nozzel, with or without coaxial additionalpresurized air (or inert gaz). Such droplets being formed in a largellyventillated area (at a temperature between +20° C. and +250° C.) so thatquick drying by evaporation process is obtained. Dry powder is obtainedafter cyclone separation.

Another preferred process according to the invention is a sprayfreezing-drying process in which tiny droplets of the composition areobtained by pumping the liquid composition in a spray nozzel, with orwithout coaxial additional of presurized air (or inert gaz), suchdroplets being formed in a very cold chamber (between −5° C. to −195.8°C.) so that quick freezing of the droplet is obtained. Drying of thisfroozen material is obtained following the standard freeze dryingprocess.

Another preferred process according to the invention is vaccum dryingprocess. Such a process comprises aseptic filling of sterile vials witha sterile filtered solution of the composition. A sterile freeze dryingrubber stopper is partially inserted into the vial, then controlledvacumm is applied on the vial so evaporation proceed until final drying.After drying the stopper is fully inserted before removing the vial fromthe drying unit.

The lyophilisation or drying process is controlled so as to ensure aresidual moisture level in the cake is below 3%, preferably below 2%,more preferably at around 1% or less. The antigen encapsulated thereinwill remain biologically active upon administration, that is to saycapable of inducing an immune response in the host.

It is foreseen that compositions of the present invention will be usedto formulate vaccines containing antigens derived from a wide variety ofsources. For example, antigens may include human, bacterial, or viralnucleic acid, pathogen derived antigen or antigenic preparations, tumourderived antigen or antigenic preparations, host-derived antigens,including GnRH and IgE peptides, recombinantly produced protein orpeptides, and chimeric fusion proteins.

Preferably the vaccine formulations of the present invention contain anantigen or antigenic composition capable of eliciting an immune responseagainst a human pathogen, which antigen or antigenic composition isderived from HIV-1, (such as Tat, nef, gp120 or gp160), human herpesviruses, such as gD or derivatives thereof or Immediate Early proteinsuch as ICP27 from HSV1 or HSV2, cytomegalovirus ((esp Human)(such as gBor derivatives thereof), Epstein Barr virus (such as gp350 orderivatives thereof), Varicella Zoster Virus (such as gpI, II and IE63),or from a hepatitis virus such as hepatitis B virus (for exampleHepatitis B Surface antigen or a derivative thereof), hepatitis A virus,hepatitis C virus and hepatitis E virus, or from other viral pathogens,such as paramyxoviruses: Respiratory Syncytial virus (such as F and Gproteins or derivatives thereof), parainfluenza virus, measles virus,mumps virus, human papilloma viruses (for example HPV6, 11, 16, 18, . .. ), flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borneencephalitis virus, Japanese Encephalitis Virus) or Influenza virus(whole live or inactivated virus, split influenza virus, grown in eggsor MDCK cells, or Vero cells or whole flu virosomes (as described by R.Gluck, Vaccine, 1992, 10, 915-920) or purified or recombinant proteinsthereof, such as HA, NP, NA, or M proteins, or combinations thereof), orderived from bacterial pathogens such as Neisseria spp, including N.gonorrhea and N. meningitidis (for example capsular polysaccharides andconjugates thereof, transferrin-binding proteins, lactoferrin bindingproteins, PilC, adhesins); S. pyogenes (for example M proteins orfragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S.mutans; H. ducreyi; Moraxella spp, including M catarrhalis, also knownas Branhamella catarrhalis (for example high and low molecular weightadhesins and invasins); Bordetella spp, including B. pertussis (forexample pertactin, pertussis toxin or derivatives thereof, filamenteoushemagglutinin adenylate cyclase, fimbriae), B. parapertussis and B.bronchiseptica; Mycobacterium spp., including M. tuberculosis (forexample ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M.paratuberculosis, M. smegmatis; Legionella spp, including L.pneumophila; Escherichia spp, including enterotoxic E. coli (for examplecolonization factors, heat-labile toxin or derivatives thereof,heat-stable toxin or derivatives thereof), enterohemorragic E. coli,enteropathogenic E. coli (for example shiga toxin-like toxin orderivatives thereof); Vibrio spp, including V. cholera (for examplecholera toxin or derivatives thereof); Shigella spp, including S.sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y.enterocolitica (for example a Yop protein), Y. pestis, Y.pseudotuberculosis; Campylobacter spp, including C. jejuni (for exampletoxins, adhesins and invasins) and C. coli; Salmonella spp, including S.typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp.,including L. monocytogenes; Helicobacter spp, including H. pylori (forexample urease, catalase, vacuolating toxin); Pseudomonas spp, includingP. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis;Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp.,including C. tetani (for example tetanus toxin and derivative thereof),C. botulinum (for example botulinum toxin- and derivative thereof), C.difficile (for example clostridium toxins A or B and derivativesthereof); Bacillus spp., including B. anthracis (for example botulinumtoxin and derivatives thereof); Corynebacterium spp., including C.diphtheriae (for example diphtheria toxin and derivatives thereof);Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA,DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (forexample OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC,DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agentof the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R.rickettsii; Chlamydia spp., including C. trachomatis (for example MOMP,heparin-binding proteins), C. pneumoniae (for example MOMP,heparin-binding proteins), C. psittaci; Leptospira spp., including L.interrogans; Treponema spp., including T. pallidum (for example the rareouter membrane proteins), T. denticola, T. hyodysenteriae; or derivedfrom parasites such as Plasmodium spp., including P. falciparum;Toxoplasma spp., including T. gondii for example SAG2, SAG3, Tg34);Entamoeba spp., including E. histolytica; Babesia spp., including B.microti; Trypanosoma spp., including T. cruzi; Giardia spp., includingG. lamblia; Leshmania spp., including L. major; Pneumocystis spp.,including P. carinii, Trichomonas spp., including T. vaginalis;Schisostoma spp., including S. mansoni, or derived from yeast such asCandida spp., including C. albicans; Cryptococcus spp., including C.neoformans. In a preferred aspect of the invention, the rapidlydissolving vaccine cake for oral administration does not compriserotavirus.

Preferred bacterial vaccines comprise antigens derived fromStreptococcus spp, including S. pneumoniae (for example capsularpolysaccharides and conjugates thereof, PsaA, PspA, streptolysin,choline-binding proteins) and the protein antigen Pneumolysin (BiochemBiophys Acta, 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25,337-342), and mutant detoxified derivatives thereof (WO 90/06951; WO99/03884). Other preferred bacterial vaccines comprise antigens derivedfrom Haemophilus spp., including H. influenzae type B (for example PRPand conjugates thereof), non typeable H. influenzae, for example OMP26,high molecular weight adhesins, P5, P6, protein D and lipoprotein D, andfimbrin and fimbrin derived peptides (U.S. Pat. No. 5,843,464) ormultiple copy varients or fusion proteins thereof. Other preferredbacterial vaccines comprise antigens derived from Morexella Catarrhalis(including outer membrane vesicles thereof, and OMP106 (WO97/41731)) andfrom Neisseria mengitidis B (including: outer membrane vesicles thereof,and NspA (WO 96/29412).

Derivatives of Hepatitis B Surface antigen are well known in the art andinclude, inter alia, those PreS1, PreS2 S antigens set forth describedin European Patent applications EP-A414 374; EP-A-0304 578, and EP198-474. In one preferred aspect the vaccine formulation of theinvention comprises the HV-1 antigen, gp120, especially when expressedin CHO cells. In a further embodiment, the vaccine formulation of theinvention comprises gD2t as hereinabove defined.

In a preferred embodiment of the present invention vaccines comprise anantigen derived from the Human Papilloma Virus (HPV) considered to beresponsible for genital warts, (HPV 6 or HPV 11 and others), and the HPVviruses responsible for cervical cancer (HPV16, HPV18 and others).

Particularly preferred forms of genital wart prophylactic, ortherapeutic, vaccine comprise L1 particles or capsomers, and fusionproteins comprising one or more antigens selected from the HPV 6 and HPV11 proteins E6, E7, L1, and L2. The most preferred forms of fusionprotein are: L2E7 as disclosed in WO 96/26277, and proteinD(1/3)-E7disclosed in GB 9717953.5 (PCT/EP98/05285).

A preferred HPV cervical infection or cancer, prophylaxis or therapeuticvaccine, composition may comprise HPV 16 or 18 antigens. For example, L1or L2 antigen monomers, or L1 or L2 antigens presented together as avirus like particle (VLP) or the L1 alone protein presented alone in aVLP or capsomer structure. Such antigens, virus like particles andcapsomer are per se known. See for example WO94/00152, WO94/20137,WO94/05792, and WO93/02184. Additional early proteins may be includedalone or as fusion proteins such as preferably E7, E2 or E5 for example;particularly preferred embodiments of this includes a VLP comprisingL1E7 fusion proteins (WO 96/11272).

Particularly preferred HPV 16 antigens comprise the early proteins E6 orE7 in fusion with a protein D carrier to form Protein D—E6 or E7 fusionsfrom HPV 16, or combinations thereof; or combinations of E6 or E7 withL2 (WO 96/26277). Alternatively the HPV 16 or 18 early proteins E6 andE7, may be presented in a single molecule, preferably a Protein D—E6/E7fusion. Such vaccine may optionally contain either or both E6 and E7proteins from HPV 18, preferably in the form of a Protein D—E6 orProtein D—E7 fusion protein or Protein D E6/E7 fusion protein.

The vaccine of the present invention may additionally comprise antigensfrom other HPV strains, preferably from strains HPV 6, 11, 31, 33, or45.

Vaccines of the present invention further comprise antigens derived fromparasites that cause Malaria. For example, preferred antigens fromPlasmodia falciparum include RTS,S and TRAP. RTS is a hybrid proteincomprising substantially all the C-terminal portion of thecircumsporozoite (CS) protein of P. falciparum linked via four aminoacids of the preS2 portion of Hepatitis B surface antigen to the surface(S) antigen of hepatitis B virus. It's full structure is disclosed inthe International Patent Application No. PCT/EP92/02591, published underNumber WO 93/10152 claiming priority from UK patent application No.9124390.7. When expressed in yeast RTS is produced as a lipoproteinparticle, and when it is co-expressed with the S antigen from HBV itproduces a mixed particle known as RTS,S. TRAP antigens are described inthe International Patent Application No. PCT/GB89/00895, published underWO 90/01496. A preferred embodiment of the present invention is aMalaria vaccine wherein the antigenic preparation comprises acombination of the RTS,S and TRAP antigens. Other plasmodia antigensthat are likely candidates to be components of a multistage Malariavaccine are P. faciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2,Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25,Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and their analogues inPlasmodium spp.

The formulations may also contain an anti-tumour antigen and be usefulfor the immunotherapeutic treatment cancers. For example, the adjuvantformulation finds utility with tumour rejection antigens such as thosefor prostrate, breast, colorectal, lung, pancreatic, renal or melanomacancers. Exemplary antigens include MAGE 1 and MAGE 3 or other MAGEantigens for the treatment of melanoma, PRAME, BAGE or GAGE (bobbins andKawakami, 1996, Current Opinions in Immunology 8, pps 628-636; Van denEynde et al., International Journal of Clinical & Laboratory Research(submitted 1997); Correale et al. (1997), Journal of the National CancerInstitute 89, p293. Indeed these antigens are expressed in a wide rangeof tumour types such as melanoma, lung carcinoma, sarcoma and bladdercarcinoma. Other Tumor-Specific antigens include, but are not restrictedto Prostate specific antigen (PSA) or Her-2/neu, KSA (GA733), MUC-1,PRAME, carcinoembryonic antigen (CEA), PSCA (PNAS 95(4) 1735-1740 1998),PSMA, P501S (sequence ID no 113 of Wo98/37814) also know as PS108 orprostein (WO 98/50567). Another tumour antigen is prostase, aprostate-specific serine protease (trypsin-like), disclosed in Ferguson,et al. (Proc. Natl. Acad. Sci. USA 1999, 96, 3114-3119) and inInternational Patent Applications No. WO 98/12302 (and also thecorresponding granted patent U.S. Pat. No. 5,955,306), WO 98/20117 (andalso the corresponding granted patents U.S. Pat. No. 5,840,871 and U.S.Pat. No. 5,786,148) (prostate-specific kallikrein) and WO 00/04149(P703P) Other prostate specific antigens are known from Wo98/37418, andWO/004149. Another antigen is STEAP PNAS 96 14523 14528 7-12 1999. Othertumour associated antigens useful in the context of the presentinvention include: Plu-1 J. Biol. Chem 274 (22) 15633-15645, 1999,HASH-2 (WO 01/62778), Cripto (Salomon et al Bioessays 199, 21 61-70,U.S. Pat. No. 5,654,140) Criptin U.S. Pat. No. 5,981,215. Additionally,antigens particularly relevant for vaccines in the therapy of canceralso comprise tyrosinase and survivin.

Other tumour-specific antigens are suitable for use in the presentinvention and include, but are not restricted to tumour-specificgangliosides such as GM 2, and GM3 or conjugates thereof to carrierproteins; or said antigen may be a self peptide hormone such as wholelength Gonadotrophin hormone releasing hormone (GNRH, WO 95/20600), ashort 10 amino acid long peptide, useful in the treatment of manycancers, or in immunocastration.

It is foreseen that compositions of the present invention will be usedto formulate vaccines containing antigens derived from Borrelia sp. Forexample, antigens may include nucleic acid, pathogen derived antigen orantigenic preparations, recombinantly produced protein or peptides, andchimeric fusion proteins. In particular the antigen is OspA. The OspAmay be a full mature protein in a lipidated form virtue of the host cell(E. Coli) termed (Lipo-OspA) or a non-lipidated derivative. Suchnon-lipidated derivatives include the non-lipidated NS1-OspA fusionprotein which has the first 81 N-terminal amino acids of thenon-structural protein (NS1) of the influenza virus, and the completeOspA protein, and another, MDP-OspA is a non-lipidated form of OspAcarrying 3 additional N-terminal amino acids.

Vaccines of the present invention may be used for the prophylaxis ortherapy of allergy. Such vaccines would comprise allergen specific (forexample Der p1) and allergen non-specific antigens (for example peptidesderived from human IgE, including but not restricted to the stanworthdecapeptide (EP 0 477 231 B1)).

Upon use but before administration, the compositions according to theinvention are generally reconstituted in a pharmaceutically acceptablediluent or carrier. Example of pharmaceutically acceptable diluentinclude water and saline. The reconstitution of the composition, or thevaccine, preferably takes place just before the administration of thecomposition to the patient. Preferably the time of dissolution of thecake is less than 10 seconds, more preferably less than 5 seconds,preferably less than 3 seconds and most preferably in less than 1second.

In some embodiments of the present invention, the antigens will beformulated with a pharmaceutical carrier. Suitable pharmaceuticalcarriers for use in the vaccine according to the invention include thoseknown in the art as being suitable for oral administration, especiallyto infants.

It may also be advantageous to formulate the virus of the invention inlipid-based vehicles such as virosomes or liposomes, in oil in wateremulsions or with carrier particles.

Further immunostimulants which may advantageously be included aresaponin derivatives such as QS21 and monophosphoryl lipid. A, inparticular 3-de-O-acylated monophosphoryl lipid A (3D-MPL). Purifiedsaponins as adjuvants are described in WO 98/56415. Saponins andmonophosphoryl lipid A may be employed separately or in combination(e.g. WO 94/00153) and may be formulated in adjuvant systems togetherwith other agents. 3D-MPL is a well-known adjuvant manufactured by RibiImmunochem, Montana and its manufacture is described in GB 2122204.

Aluminium hydroxide is a particularly preferred component of a vaccinecomposition according to the invention.

Suitably a solution of the antigenic composition according to theinvention and obtained after reconstitution is an isotonic solution. Ina preferred embodiment the pH of the composition of the invention isfrom about 6 to about 10, preferably from about 7 to about 9. Theantigenic composition of the invention when reconstituted isadministered mucosally or parenterally, preferably parenterally, mostpreferably by injection intravenously, subcutaneously orintramuscularly. However, such formulations may be sought in anotherappropriate dosage form if necessary (e.g. nasal sprays and othermucosal forms).

The compositions according to the invention when for parenteraladministration, may be packed in suitably adapted pharmaceuticalapplication devices, for example syringes, vials or ampoules, such thatthe addition of the diluent or carrier allows in situ preparation of anaqueous solution of the active ingredient in a form suitable forimmediate administration to the patient. Such devices form a furtheraspect of the invention. When for mucosal administration, lyophilised ordried formulations may conveniently be provided in the form of tabletsin a pharmaceutical blister pack.

In another aspect the invention provides a composition comprising a liveattenuated bacterium or virus, or live viral or bacterial vector,wherein the composition is a lyophilised solid or a dried solidaccording to the invention, capable of immediate dissolution whenadministered to the recipient.

Vaccines of the invention may be formulated and administered by knowntechniques, using a suitable amount of live virus to provide effectiveprotection against infection without significant adverse side effects intypical vaccinees. A suitable amount of live virus will normally bebetween 10⁴ and 10⁷ ffu per dose. A typical dose of vaccine may comprise10⁵-10⁶ ffu per dose and may be given in several doses over a period oftime, for example in two doses given with a two-month interval. Benefitsmay however be obtained by having more than 2 doses, for example a 3 or4 dose regimen, particularly in developing countries. The intervalbetween doses may be more or less than two months long. An optimalamount of live virus for a single dose or for a multiple dose regimen,and optimal timing for the doses, can be ascertained by standard studiesinvolving observation of antibody titres and other responses insubjects.

The amount of protein in each vaccine dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccinees. Such amount will vary depending uponwhich specific immunogen is employed and how it is presented. Generally,it is expected that each dose will comprise 1-1000 μg of protein,preferably 1-500 μg, preferably 1-100 μg, most preferably 1 to 50 μg. Anoptimal amount for a particular vaccine can be ascertained by standardstudies involving observation of appropriate immune responses insubjects. Following an initial vaccination, subjects may receive one orseveral booster immunisations adequately spaced.

The present invention is illustrated by the following examples.

EXAMPLE I

Maximum PO₄—NaCl Buffer Concentration Giving an Acceptable LyophilisedCake, Using Standard Lyophilisation Sugars

1.1. Introduction

Various phosphate (PO₄)/NaCl buffer concentrations were submitted to astandard lyophilisation cycle.

The Phosphate—NaCl buffer was compared to a simple phosphate buffer(free of NaCl).

In those preparations, concentrations were adjusted in order to havecomparable iso-osmotic pressure across each buffer for similar dilution(including osmotic pressure contribution of carbohydrate). In all tablespresented hereafter, the concentration present in the final formulationjust before the lyophilisation step is reported. PO₄/NaCl PO₄ 8/120 mM120 mM  6/90 mM 90 mM 4/60 mM 60 mM 2/30 mM 30 mM 1/15 mM 15 mM1.2. Results

The aspect of the cake is assessed according to the criteria listedExample 3.2.

1.2.1. Sucrose (3.15%) Compositions cake cake PO₄/ mOsm/ cake at t = 1 Wat t = 2 W NaCl Kg at t = 0 37° C. 37° C. Results 8-120 mM  297 no cakeno cake no cake Fail 6-90 mM 268 no cake no cake no cake Fail 4-60 mM204 retracted Retracted Retracted Fail 2-30 mM 153 slightly slightlyslightly Pass retracted retracted retracted 1-15 mM 124 slightlyslightly slightly Pass retracted retracted retracted

cake cake mOsm/ cake at t = 1 W at t = 2 W PO₄ Kg at t = 0 37° C. 37° C.Results 120 mM  352 slightly retracted retracted Fail retracted 90 mM299 slightly slightly slightly Pass retracted retracted retracted 60 mM218 slightly slightly slightly Pass retracted retracted retracted 30 mM160 slightly slightly slightly Pass retracted retracted retracted 15 mM127 slightly slightly slightly Pass retracted retracted retracted

1.2.2. Maltose (3.15%) Compositions mOsm/ cake cake T = 1 cake T = 2PO₄—NaCl kg T = 0 W 37° C. W 37° C. Results 8-120 mM  299 no cake nocake no cake Fail 6-90 mM 267 retracted retracted retracted Fail 4-60 mM206 retracted retracted retracted Fail 2-30 mM 145 slightly slightlyslightly Pass retracted retracted retracted 1-15 mM 113 slightlyslightly slightly Pass retracted retracted retracted

mOsm/ cake cake T = 1 cake T = 2 PO₄ kg T = 0 W 37° C. W 37° C. Results120 mM  334 slightly retracted retracted Fail retracted 90 mM 272slightly retracted retracted Fail retracted 60 mM 215 slightly slightlyslightly Pass retracted retracted retracted 30 mM 153 slightly slightlyslightly Pass retracted retracted retracted 15 mM 117 nice cake slightlyslightly Pass retracted retracted

1.2.3. Trehalose (3.15%) Compositions mOsm/ cake cake T = 1 cake T = 2PO₄—NaCl kg T = 0 W 37° C. W 37° C. Results 8-120 mM  312 no cake nocake no cake Fail 6-90 mM 265 no cake no cake no cake Fail 4-60 mM 204retracted retracted retracted Fail 2-30 mM 142 slightly slightlyslightly Pass retracted retracted retracted 1-15 mM 112 slightlyslightly slightly Pass retracted retracted retracted

mOsm/ cake cake T = 1 cake T = 2 PO₄ kg T = 0 W 37° C. W 37° C. Results120 mM  326 retracted retracted retracted Fail 90 mM 293 retractedretracted retracted Fail 60 mM 215 slightly slightly slightly Passretracted retracted retracted 30 mM 146 slightly slightly slightly Passretracted retracted retracted 15 mM 111 slightly slightly slightly Passretracted retracted retracted

1.2.4. Raffinose (3.15%) Compositions mOsm/ cake cake T = 1 cake T = 2PO₄—NaCl kg T = 0 W 37° C. W 37° C. Results 8-120 mM  288 no cake nocake no cake Fail 6-90 mM 232 retracted retracted retracted Fail 4-60 mM168 slightly retracted retracted Fail retracted 2-30 mM 111 slightlyslightly slightly Pass retracted retracted retracted 1-15 mM 70 slightlyslightly slightly Pass retracted retracted retracted

mOsm/ cake cake T = 1 cake T = 2 PO₄ kg T = 0 W 37° C. W 37° C. Result120 mM  289 slightly slightly slightly Pass retracted retractedretracted 90 mM 231 slightly slightly slightly Pass retracted retractedretracted 60 mM 173 slightly slightly slightly Pass retracted retractedretracted 30 mM 118 slightly slightly slightly Pass retracted retractedretracted 15 mM 80 slightly slightly slightly Pass retracted retractedretracted

1.2.5. CONCLUSIONS

The presence of Phosphate (PO₄)/NaCl in formulation is not compatiblewith lyophilisation. Indeed, the resulting cake structure is notacceptable when a concentration higher than 2 mM PO₄/30 mM NaCl is used.Sucrose, maltose, trehalose and raffinose give the same results whenusing the PO₄/NaCl buffer.

NaCl is clearly responsible of the bad results during lyophilisation.Indeed, much higher concentrations can be reached with PO₄ (free ofNaCl) buffer:

-   -   up to 120 mM PO₄ with raffinose    -   up to 90 mM PO₄ in sucrose    -   up to 60 mM PO₄ for maltose and trehalose        1.3. Artistic View of Sucrose Molecular Arrangement

FIG. 2 shows an artistic view of amorphous sucrose molecular arrangementin the absence of NaCl. The remaining water molecules are not shown.

The lyophilised cake structure is build essentially on the“sucrose-sucrose” molecular interactions. Those intermolecularinteractions are the results of molecular orientation, molecularconformation and atomic radii of the constituent atoms.

Due to the amorphous structure (high degree of desorder betweenmolecules), intermolecular interactions are weaker compared to thoseusually found in a crystalline structure (high degree of order betweenmolecules).

By comparison, FIG. 3 shows an artistic view of amorphous moleculararrangement of about 90 mM sucrose in presence of 60 mM NaCl. Theremaining water molecules are not shown.

The “sucrose-sucrose” molecular interactions are perturbed by the ionicinteraction of sodium chloride. The experimental results shown inExample I that the presence of NaCl decreases the “sugar-sugar”intermolecular interactions.

EXAMPLE II

Assessment of Several Carbohydrates (or Excipient) Leading to anAcceptable Lyophilised Cake at a Given Fixed PO₄ (4 mM)—NaCl (60 mM)Concentration

The target value for these experiments has been set at a concentrationof PO₄ (6 mM)—NaCl (60 mM), a concentration of NaCl which, as shown inexample I, leads to an unacceptable lyophilised cake structure, andwhich is often reached in a final antigenic solution after purification.

EXPERIMENT 1

Consolidation of the Cake Structure by Using Carbohydrate MoleculesExhibiting a Carboxylic Function, in Addition to Conventional Sucrose.

Lactobionic acid is illustrated in FIG. 1. Carbo- PO₄/ hydrate Cake at 1Re- Sucrose NaCl derivative mM Cake w 37° C. sult 2.08% 4 mM/ Ca- 9.6 mMSlightly Slightly Pass 60 mM lactobionate retracted re- tracted

EXPERIMENT 2

Consolidation of the Cake Structure by Using Carbohydrate MoleculesExhibiting a Carboxylic Function in the Absence of Sucrose.

Thus, in the lactobionate series, the nature of the salt (Na, Ca, orTris counter-ion) plays a capital role in the “sugar-sugar”interactions. Tris molecule is bigger (compared to Na), and so ispushing “carbohydrate” molecules far away from each other, reducing the“sugar-sugar” interaction (compared to Na).

At fixed NaCl concentrations: Carbo- Carbo- PO₄/ hydrate hydrate CakeCake NaCl derivative mM at T = 0 at 1 W 37° C. Results 4 mM/ Na 87 OK OKPass 60 mM lactobionate 4 mM/ Ca 87 Slightly Slightly Pass 60 mMlactobionate retracted retracted

At decreasing NaCl concentrations: Car- bo- Carbohy- hy- PO₄/ dratedrate MOs Cake Cake at 1 NaCl derivative mM m kg at T = 0 W 37° C.Result 4 mM/ Na 87 242 Slightly Slightly Pass 60 mM Lactobionateretracted retracted 4 mM/ Na 80 251 Slightly Slightly Pass 60 mMLactobionate retracted retracted 0.8 mM/ Na 70 143 Cake OK Cake OK Pass12 mM Lactobionate 1.6 mM/ Na 70 175 Slightly Slightly Pass 24 mMLactobionate retracted retracted 2.4 mM/ Na 70 197 Slightly SlightlyPass 36 mM Lactobionate retracted retracted 3.2 mM/ Na 70 219 SlightlySlightly Pass: 48 mM Lactobionate retracted retracted limit

EXPERIMENT 3

Consolidation of the Cake Structure by Using Carbohydrate MoleculesExhibiting a Carboxylic Function, Using Different Carboxylic AcidDerivatives of Carbohydrates. Cake PO₄/ Carbohydrate Carbohydrate Na mOsCake At 7 W NaCl derivative salt mM m/kg At T = 0 37° C. Result 4 mM/Galacturonic 90 mM 272 Slightly Slightly Pass 60 mM retracted retracted4 mM/ Galacturonic 85 mM 265 Slightly Slightly Pass 60 mM retractedretracted 4 mM/ Galacturonic 80 mM 254 Slightly Slightly Limit 60 mMretracted retracted 4 mM/ Galacturonic 80 mM 254 Slightly Slightly Pass60 mM retracted retracted 4 mM/ Lactobionic 100 mM  280 SlightlySlightly Pass 60 mM retracted retracted 4 mM/ Lactobionic 95 mM 268Slightly Slightly Pass 60 mM retracted retracted 4 mM/ Lactobionic 90 mM263 Slightly Slightly Limit 60 mM retracted retracted 4 mM/Na-Lactobionate 92 mM 277 Slightly Slightly Pass 60 mM retractedretracted 4 mM/ Na-Galacturonate 92 mM 247 Slightly Slightly Pass 60 mMretracted retracted

EXAMPLE III

Material and Methods

1. Preparation of the Antigenic Composition

The formulated bulk mix is prepared by asepticly adding and mixing stepby step successively the correct quantities of sterile water, sterileconcentrated carbohydrate solutions, sterile concentrated buffersolutions, sterile concentrated salt solutions, and sterile concentratedantigen solutions.

The formulated bulk mix is distributed in the vials for lyophilisation(typically 3 ml siliconed glass vials filled with 0.5 ml of formulatedbulk).

Lyophilisation rubber stoper is placed on vials (partially closed).

2. Lyophilisation

The vials containing (0.5 ml) of formulated vaccine were placed inside aprecooled (−69° C.) lyophilisator (Martin-Christ or Usifroid), whilstthe lyophilisation conditions were monitored and adjusted in order tofollow as close as possible the following time-temperature curve:

At the end of the processes under vacuum, the vials were closed bypushing the stopper completely.

Then stopper are sealed by aluminium capsules.

Lyophilised vials are storred at at +4° C. until analysis orutilisation.

3. Inspection of the Cake Aspect

3.1. Accelerated Stability at 37° C.

Ten lyophilised vials of each formulation are placed for a period of oneweek in an ventilated oven regulated at 37° C., then stored at +4° C.The cake is then inspected.

3.2. Cake Inspection

Lyophilised cakes are inspected visually just after lyophilisation andafter accelerated stability testing (7 days at 37° C.).

The percentage of retraction is estimated by size measurement of thecakes, and classified according to one of the different categories:

-   -   Retracted means that initial size of the cake (height or        diameter) is reduced at least by 20%    -   Slightly retracted means that the initial size of the cake        (height or diameter) is reduced by a factor of at most 20%    -   Pass means that the initial size of the cake (height or        diameter) is reduced not more that 20% just after lyophilisation        and also after 1 week at 37° C. and also that not color change        occurred after 1 week at 37° C.    -   Fail means that the initial size of the cake (height or        diameter) is reduced at least by 20% just after lyophilisation        or that after 1 week at 37° C. or that a color change is ccurred        after 1 week at 37° C.

Measurement of the cake side is done applying a graduated rular on thebottom of the vials and measuring the cake diametre (or height) throughthe glass.

4. Buffers

Phosphate-NaCl buffer also known in house as the “Phosphate 10-150” ischosen because most of antigens are purified in this buffer. It shouldbe pointed out that we used the NaH₂PO₄+K₂HPO₄ combination. The selectedpH value is obtained by weighting the correct NaH₂PO₄/K₂HPO₄ proportion.Thus, no HCl or NaOH is used to adjust the pH of the buffer.

Decreasing quantities: 0.4, 0.3, 0.2, 0.1 and 0.05 ml of buffer are usedduring formulation. Final filling volume being always 0.5 ml.

Phosphate buffer free of NaCl is chosen to be compared to thephosphate-NaCl buffer. Starting with a 150 mM solution, decreasingquantities: 0.4, 0.3, 0.2, 0.1 and 0.05 ml of buffer are used duringformulation.

Osmotic Pressure

Measurements were performed on a Friske Type one-ten osmometer.

1. An antigenic composition suitable for freeze-drying, spray-drying,spray freezing-drying or vacuum drying comprising an antigen within anaqueous medium, a carbohydrate, and optionally a preservative, whereinthe carbohydrate is in the form of a derivative which exhibit acidicmoieties and wherein the aqueous medium comprises NaCl concentration ofabove 15 mM.
 2. An antigenic composition as claimed in 1, wherein thecarbohydrate is in the form of its carboxylic acid derivative.
 3. Anantigenic composition as claimed in 2, wherein the carboxylic derivativeis selected from the list consisting of lactobionic acid, gluconic acid,glucuronic acid, galacturonic acid or galactaric acid.
 4. An antigeniccomposition as claimed in claim 1, wherein the aqueous medium contains aNaCl concentration of at least 30 mM.
 5. An antigenic composition asclaimed in claim 1 which is in spray-dried, vacuum-dried or freeze-driedform.
 6. An antigenic composition as claimed in claim 5 which is inreconstituted form.
 7. An antigenic composition as claimed in claim 1,wherein the reconstituted composition comprises an adjuvant.
 8. Anantigenic composition as claimed in claim 7, wherein the adjuvant isselected from: oil-in-water emulsion, 3D-MPL, CpG, QS21 or a mixture oftwo or more thereof.
 9. An antigenic composition as claimed in claim 1,wherein the antigen or antigen composition is derived from the groupcomprising: Human Imunodeficiency Virus, Varicella Zoster virus, HerpesSimplex Virus type 1, Herpes Simplex virus type 2, Humancytomegalovirus, Dengue virus, Hepatitis A, B, C or E, RespiratorySyncytial virus, human papilloma virus, Influenza virus, Hib, Meningitisvirus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella,Plasmodium or Toxoplasma, stanworth decapeptide; or Tumour associatedantigens (TMA), MAGE, BAGE, GAGE, MUC-1, Her-2 neu, LnRH, CEA, PSA, KSA,or PRAME, Cripto, HASH2, prostase, prostein.
 10. A process for thepreparation of an antigenic composition according to claim 1, comprisingmixing the ingredients of the composition and subjecting the mixture toa lyophilisation, vacuum drying or spray drying procedure.
 11. A processfor the preparation of an antigenic composition according to claim 4,comprising mixing the ingredients of the composition and either freezingthem and drying the frozen mixture, or spraying them.